Igf-1 receptor interacting proteins

ABSTRACT

The invention comprises a nucleic acid molecule with the sequence SEQ ID NO:5 and the complementary sequence, and its use in diagnosis and therapy. This nucleic acid molecule (IIP-10) is a gene which encodes an IGF-1 receptor binding polypeptide.

PRIORITY TO RELATED APPLICATIONS

This application is a Continuation of application Ser. No. 10/867,045,filed Jun. 14, 2004 which is now allowed, which is a Divisional ofapplication Ser. No. 09/917,974, filed Jul. 30, 2001 which is nowallowed, which is a Divisional of copending application Ser. No.09/453,195, filed Dec. 2, 1999, now U.S. Pat. No. 6,368,826; whichclaims the benefit of European Application No. 98122992.5, filed Dec. 3,1998. The entire contents of the above-identified applications arehereby incorporated by reference.

BACKGROUND OF THE INVENTION

The IGF-1 receptor signaling system plays an important role in tumorproliferation and survival and is implicated in inhibition of tumorapoptosis. In addition and independent of its mitogenic properties,IGF-1R activation can protect against or at least retard programmed celldeath in vitro and in vivo (Harrington et al., EMBO J. 13 (1994)3286-3295; Sell et al., Cancer Res. 55 (1995) 303-305; Singleton et al.,Cancer Res. 56 (1996) 4522-4529). A decrease in the level of IGF-1Rbelow wild type levels was also shown to cause massive apoptosis oftumor cells in vivo (Resnicoff et al., Cancer Res. 55 (1995) 2463-2469;Resnicoff et al., Cancer Res. 55 (1995) 3739-3741). Overexpression ofeither ligand (IGF) and/or the receptor is a feature of various tumorcell lines and can lead to tumor formation in animal models.Overexpression of human IGF-1R resulted in ligand-dependentanchorage-independent growth of NIH 3T3 or Rat-1 fibroblasts andinoculation of these cells caused a rapid tumor formation in nude mice(Kaleko et al., Mol. Cell. Biol. 10 (1990) 464-473; Prager et al., Proc.Natl. Acad. Sci. USA 91 (1994) 2181-2185). Transgenic miceoverexpressing IGF-II specifically in the mammary gland develop mammaryadenocarcinoma (Bates et al., Br. J. Cancer 72 (1995) 1189-1193) andtransgenic mice overexpressing IGF-II under the control of a moregeneral promoter develop an elevated number and wide spectrum of tumortypes (Rogler et al., J. Biol. Chem. 269 (1994) 13779-13784). Oneexample among many for human tumors overexpressing IGF-I or IGF-II atvery high frequency (>80%) are Small Cell Lung Carcinomas (Quinn et al.,J. Biol. Chem. 271 (1996) 11477-11483). Signaling by the IGF systemseems to be also required for the transforming activity of certainoncogenes. Fetal fibroblasts with a disruption of the IGF-1R gene cannotbe transformed by the SV40 T antigen, activated Ha-ras, or a combinationof both (Sell et al., Proc. Natl. Acad. Sci. USA 90 (1993) 11217-11221;Sell et al., Mol. Cell. Biol. 14 (1994) 3604-3612), and the E5 proteinof the bovine papilloma virus is also no longer transforming (Morrioneet al., J. Virol. 69 (1995) 5300-5303). Interference with the IGF/IGF-1Rsystem was also shown to reverse the transformed phenotype and toinhibit tumor growth (Trojan et al., Science 259 (1993) 94-97; Kalebicet al., Cancer Res. 54 (1994) 5531-5534; Prager et al., Proc. Natl.Acad. Sci. USA 91 (1994) 2181-2185; Resnicoff et al., Cancer Res. 54(1994) 2218-2222; Resnicoff et al., Cancer Res. 54 (1994) 4848-4850;Resnicoff et al., Cancer Res. 55 (1995) 2463-2469. For example, miceinjected with rat prostate adenocarcinoma cells (PA-III) transfectedwith IGF-1R antisense cDNA (729 bp) develop tumors 90% smaller thancontrols or remained tumor-free after 60 days of observation (Burfeindet al., Proc. Natl. Acad. Sci. USA 93 (1996) 7263-7268). IGF-1R mediatedprotection against apoptosis is independent of de-novo gene expressionand protein synthesis. Thus, IGF-1 likely exerts its anti-apoptoticfunction via the activation of preformed cytosolic mediators.

Some signaling substrates which bind to the IGF-1R (e.g. IRS-1, SHC, p85PI3 kinase etc., for details see below) have been described. However,none of these transducers is unique to the IGF-1R and thus could beexclusively responsible for the unique biological features of the IGF-1Rcompared to other receptor tyrosine kinase including the insulinreceptor. This indicates that specific targets of the IGF-1R (or atleast the IGF-receptor subfamily) might exist which trigger survival andcounteract apoptosis and thus are prime pharmaceutical targets foranti-cancer therapy.

By using the yeast two-hybrid system it was shown that p85, theregulatory domain of phosphatidyl inositol 3 kinase (PI3K), interactswith the IGF-1R (Lamothe, B., et al., FEBS Lett. 373 (1995) 51-55;Tartare-Decker, S., et al., Endocrinology 137 (1996) 1019-1024). Howeverbinding of p85 to many other receptor tyrosine kinases of virtually allfamilies is also seen. Another binding partner of the IGF-1R defined bytwo-hybrid screening is SHC which binds also to other tyrosine kinasessuch as trk, met, EGF-R and the insulin receptor (Tartare-Deckert, S.,et al., J. Biol. Chem. 270 (1995) 23456-23460). The insulin receptorsubstrate 1 (IRS-1) and insulin receptor substrate 2 (IRS-2) were alsofound to both interact with the IGF-1R as well as the insulin receptor(Tartare-Deckert, S., et al., J. Biol. Chem. 270 (1995) 23456-23460; He,W., et al., J. Biol. Chem. 271 (1996) 11641-11645; Dey, R. B., et al.,Mol. Endocrinol. 10 (1996) 631-641). Grb 10 which interacts with theIGF-1R also shares many tyrosine kinases as binding partners, e.g. met,insulin receptor, kit and abl (Dey, R. B., et al., Mol. Endocrinol. 10(1996) 631-641; Morrione, A., et al., Cancer Res. 56 (1996) 3165-3167).The phosphatase PTP1D (syp) shows also a very promiscuous bindingcapacity, i.e. binds to IGF-1R, insulin receptor, met and others(Rocchi, S., et al., Endocrinology 137 (1996) 4944-4952). More recently,mSH2-B and vav were described as binders of the IGF-1R, but interactionis also seen with other tyrosine kinases, e.g. mSH2-B also bind to retand the insulin receptor (Wang, J., and Riedel, H., J. Biol. Chem. 273(1998) 3136-3139). Taken together, the so far described IGF-1R bindingproteins represent relatively unspecific targets for therapeuticapproaches, or are in the case of the insulin receptor substrates(IRS-1, IRS-2) indispensable for insulin-driven activities.

SUMMARY OF THE INVENTION

The present invention relates to IGF-1 receptor interacting proteins(IIPs); nucleic acids coding therefor; and their use for diagnostics andtherapeutics, especially in the field of cancer. In particular, theinvention relates to the identification of said genes in mammaliancells, especially in malignant tumor cells; to gene therapy methods forinhibiting the interaction between IGF-1 receptor and IIPs; methods ofscreening for potential cancer therapy agents; and cell lines and animalmodels useful in screening for and evaluating potentially usefulpharmaceutical agents that inhibit the interaction between IIPs andIGF-1 receptor.

The present invention relates in particular to the cloning andcharacterization of the gene IIP-10 and the gene products thereof. Saidgene products (polypeptides, mRNA) are especially characterized ashaving the ability to modulate the IGF-1 receptor signaling pathway. Thefunction of the gene products according to the invention is therefore tomodulate signal transduction of the IGF-1 receptor. Forced activation ofIIPs therefore correlates with increased tumor cell proliferation,survival and escape of apoptosis.

It is an object of the invention to provide novel genes encoding bindingproteins of IGF-1R as well as the corresponding polypeptides whichmodulate, preferably activate the IGF-1 receptor signaling pathway. Itis envisioned that this invention provides a basis for new cancertherapies based on the modulation, preferably inhibition, of theinteraction between IGF-1R and IIPs.

DESCRIPTION OF THE FIGURES AND SEQUENCES

FIG. 1 Domain structure of yeast two-hybrid baits which were used toscreen cDNA libraries for cytoplasmic binding proteins of the IGF-1receptor.

The LexA DNA binding domain was fused to the cytoplasmic (cp) domain (nt2923 to 4154) of the wildtype IGF-1 receptor (a) or the kinase inactivemutant (K/A mutation at aa 1003) (b) (Ullrich, A., et al., EMBO J. 5(1986) 2503-2512; Weidner, K. M., et al., Nature 384 (1996) 173-176).The nucleotide and amino acid sequence of two different linkers insertedbetween the LexA DNA-binding domain and the receptor domain are shownbelow. The I1 (wt IGF-1 receptor) and K1 (kinase inactive mutant IGF-1receptor) constructs contain an additional proline and glycine comparedto the I2 and K2 constructs.

FIG. 2 Modification of the yeast two-hybrid LexA/IGF-1 receptor baitconstruct.

Schematic illustration of cytoplasmic binding sites of the IGF-1receptor. The α-subunits of the IGF-1 receptor are linked to theβ-chains via disulfide bonds. The cytoplasmic part of the β-chaincontains binding sites for substrates in the juxtamembrane andC-terminal domain.

Domain structure of the two-hybrid bait containing only thejuxtamembrane IGF-1 receptor binding sites. The juxtamembrane domain ofthe IGF-1 receptor (nt 2923 to 3051) (Ullrich, A., et al., EMBO J. 5(1986) 2503-2512) was fused to the kinase domain of tprmet (nt 3456 to4229) (GenBank accession number: HSU19348).

Domain structure of the two-hybrid bait containing only the C-terminalIGF-1 receptor binding sites. The C-terminal domain of the IGF-1receptor (nt 3823 to 4149) (Ullrich, A., et al., EMBO J. 5 (1986)2503-2512) was fused to the kinase domain of tprmet (nt 3456 to 4229)(GenBank accession number: HSU19348).

FIG. 3 Isoforms of IIP-1.

Delineation of the cDNA sequences of IIP-1 and IIP-1 (p26). Nucleotidesare numbered above. The potential translation initiation site within theIIP-1 cDNA is at position 63. The first ATG as potential translationinitiation site in the alternative splice variant IIP-1 (p26) is atposition 353. Both cDNAs contain a stop codon at position 1062.

Domain structure of IIP-1 and IIP-1 (p26). Amino acid positions areindicated above. In comparison to IIP-1 (p26) IIP-1 contain additional97 amino acids at the N-terminus. Both isoforms of IIP-1 contain a PDZdomain spanning a region between amino acids 129 and 213.

FIG. 4 Delineation of the IGF-1 receptor binding domain of IIP-1.

Full-length IIP-1, its partial cDNA clones (IIP-1a and IIP-1b) anddeletion mutants (IIP-1a/mu1, IIP-1a/mu2, IIP-1a/mu3, IIP-1b/mu1) wereexamined for interaction with the IGF-1 receptor in the yeast two-hybridsystem. Yeast cells were cotransfected with a LexA IGF-1 receptor fusionconstruct and an activation plasmid coding for IIP-1 or the differentIIP-1 mutants fused to the VP16 activation domain. Interaction betweenIIP-1 or its mutants and the IGF-1 receptor was analyzed by monitoringgrowth of yeast transfectants plated out on histidine deficient mediumand incubated for 6d at 30° C. (diameter of yeast colonies: +++, >1 mmin 2d; ++, >1 mm in 4d; +, >1 mm in 6d; −, no detected growth). The PDZdomain can be defined as essential and sufficient for mediating theinteraction with the IGF-1 receptor. Nucleotide positions with respectto full length IIP-1 are indicated above.

FIG. 5 Protein sequence motifs of IIP-10.

The amino acid sequence of IIP-10 was analyzed using the computerprogram “Motifs” (Wisconsin Package Version 10.0, Genetics ComputerGroup (GCG), Madison, Wis.) which looks for protein motifs by searchingprotein sequences for regular expression patterns described in thePROSITE Dictionary.

SEQ ID NO:1 Nucleotide sequence of IIP-1 (cDNA).

SEQ ID NO:2 Predicted amino acid sequence of IIP-1.

SEQ ID NO:3 Nucleotide sequence of the IIP-6 partial cDNA clone.

SEQ ID NO:4 Deduced amino acid sequence of the IIP-6 partial cDNA clone.Cysteine and histidine residues of the two Cys₂His₂ Zinc finger domainsare amino acids 72, 75, 88, 92, 100, 103, 116, and 120.

SEQ ID NO:5 Nucleotide sequence of IIP-10 (cDNA).

SEQ ID NO:6 Deduced amino acid sequence of IIP-10.

SEQ ID NO:7 Primer TIP2c-s.

SEQ ID NO:8 Primer TIP2b-r.

SEQ ID NO:9 Primer Hethyl-s.

SEQ ID NO:10 Primer Hethyl-r.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to IGF-1 receptor interacting proteins(IIPs); nucleic acids coding therefor; and their use for diagnostics andtherapeutics, especially in the field of cancer. The inventionpreferably comprises a nucleic acid encoding a protein (IIP-10) bindingto IGF-1 receptor selected from the group comprising the nucleic acidsshown in SEQ ID NO:5 or a nucleic acid sequence which is complementarythereto, nucleic acids which hybridize under stringent conditions withone of the nucleic acids from a) which encode a polypeptide showing atleast 75% homology with the polypeptide of SEQ ID NO:6 or sequencesthat, due to the degeneracy of the genetic code, encode IIP-10polypeptides having the amino acid sequence of the polypeptides encodedby the sequences of a) and b).

The cDNA of IIP-10 codes for a new protein of 226 amino acid with acalculated molecular weight of 25.697. IIP-10 is a lysine rich protein(11%). IIP-10 contains an N-glycosylation site, several N-myristoylationsites, Ck2 and PKC phosphorylation sites, one tyrosine kinasephosphorylation site and one putative nuclear localization signal (FIG.5). The cDNA sequence of IIP-10 shows 65% homology to the cDNA sequenceof the Gallus Gallus thymocyte protein cthy28kD (EMBL accession number:GG34350). The amino acid sequences of IIP-10 and cthy28kD show 70%identity. Nt 383 to nt 584 of the IIP-10 cDNA are 94% identical to apartial cDNA described in WO 95/14772 (human gene signature HUMGS06271;accession number T24253). By immunofluorescence, flag-tagged IIP-10shows both a cytoplasmic and a nuclear localization in NIH3T3 cellsoverexpressing the IGF-1 receptor. Further yeast two-hybrid analysisrevealed that IIP-10 interacts in a phosphorylation dependent mannerwith the IGF-1 receptor. IIP-10 does not interact with the insulinreceptor. Deletion analysis of IIP-10 revealed that aa 19 to aa 226 aresufficient for binding to the IGF-1 receptor.

“Interaction” or “binding between IIP10 and the IGF-1 receptor” means aspecific binding of the IIP10 polypeptide to the IGF-1 receptor but notto control proteins such as lamin in the yeast two hybrid system.Specific binding to the IGF-1 receptor can be demonstrated usingglutathion-S-transferase (GST)-IIP fusion proteins expressed in bacteriaand IGF-1 receptors expressed in mammalian cells. Furthermore, anassociation between a Flag tagged IIP-10 fusion protein (cf Weidner, K.M. et al., Nature 384 (1996) 173-176) and the IGF-1 receptor can bemonitored in mammalian cell systems. For this purpose eukaryoticexpression vectors are used to transfect the respective cDNAs.Interaction between the proteins is visualized by coimmunoprecipitationexperiments or subcellular localization studies using anti-Flag oranti-IGF-1 receptor antibodies.

Further provided by the invention are probes and primers for the genesaccording to the invention, as well as nucleic acids which encodeantigenic determinants of the gene products according to the invention.Therefore, preferred embodiments include nucleic acids with preferably10 to 50, or more preferably, 10 to 20 consecutive nucleotides out ofthe disclosed sequences.

The term “nucleic acid” denotes a polynucleotide which can be, forexample, a DNA, RNA, or derivatized active DNA or RNA. DNA and mRNAmolecules are preferred, however.

The term “hybridize under stringent conditions” means that two nucleicacid fragments are capable of hybridization to one another understandard hybridization conditions described in Sambrook et al.,Molecular Cloning: A laboratory manual (1989) Cold Spring HarborLaboratory Press, New York, USA.

More specifically, “stringent conditions” as used herein refers tohybridization in 5.0×SSC, 5×Denhardt, 7% SDS, 0.5 M phosphate buffer pH7.0, 10% dextran sulfate and 100 μg/ml salmon sperm DNA at about 50°C.-68° C., followed by two washing steps with 1×SSC at 68° C. Inaddition, the temperature in the wash step can be increased from lowstringency conditions at room temperatures, about 22° C., to highstringency conditions at about 68° C.

The invention further comprises recombinant expression vectors which aresuitable for the expression of IIP-10, recombinant host cellstransfected with such expression vectors, as well as a process for therecombinant production of a protein which is encoded by the IIP-10 gene.

The invention further comprises synthetic and recombinant polypeptideswhich are encoded by the nucleic acids according to the invention, andpreferably encoded by the DNA sequence shown in SEQ ID NO:5 as well aspeptidomimetics based thereon. Such peptidomimetics have a high affinityfor cell membranes and are readily taken up by the cells.Peptidomimetics are preferably compounds derived from peptides andproteins, and are obtained by structural modification using unnaturalamino acids, conformational restraints, isosterical placement,cyclization, etc. They are based preferably on 24 or fewer, preferably20 or fewer, amino acids, a basis of approximately 12 amino acids beingparticularly preferred.

The polypeptides and peptidomimetics can be defined by theircorresponding DNA sequences and by the amino acid sequences derivedtherefrom. The isolated IIP polypeptide can occur in natural allelicvariations which differ from individual to individual. Such variationsof the amino acids are usually amino acid substitutions. However, theymay also be deletions, insertions or additions of amino acids to thetotal sequence leading to biologically active fragments. The IIP proteinaccording to the invention—depending, both in respect of the extent andtype, on the cell and cell type in which it is expressed—can be inglycosylated or non-glycosylated form. IIP-Polypeptides with tumoricidicand/or metastatic activity can be identified by a tumor progressionassay using mammalian cells expressing said polypeptides and measuringthe proliferation capacity and apoptosis in relation to mammalian cellsnot expressing said polypeptides.

“Polypeptide with IIP-10 activity or IIP-10” therefore means proteinswith minor amino acid variations but with substantially the sameactivity as IIP-10. Substantially the same means that the activities areof the same biological properties and the polypeptides show at least 75%homology (identity) in amino acid sequences with IIP-10. Morepreferably, the amino acid sequences are at least 90% identical.Homology according to the invention can be determined with the aid ofthe computer programs Gap or BestFit (University of Wisconsin; Needlemanand Wunsch, J. Biol. Chem. 48 (1970) 443-453; Smith and Waterman, Adv.Appl. Math. 2 (1981) 482-489).

Other IIPs according to, and used by, the invention are in particular:

IIP-1

A cDNA encoding an IGF-1 receptor interacting protein which was namedIIP-1 (SEQ ID NO:1) was isolated. The cDNA of IIP-1 codes for a newprotein of 333 aa with a calculated molecular weight of 35,727. IIP-1 isa glycine rich protein (13%). IIP-1 contains several N-myristoylationsites, PKC and Ck2 phosphorylation sites and two putative nuclearlocalization signals. A second isoform, IIP-1 (p26), of 236 aa in lengthwith a calculated molecular weight of 26,071 was identified which wasgenerated most likely by alternative splicing (FIG. 3). Both isoformsbind to the IGF-1 receptor.

cDNA sequences of IIP-1 have been reported previously (Database EMBLNos. AF089818 and AF061263; DeVries, L., et al., Proc. Natl. Acad. Sci.USA 95 (1998) 12340-12345). Two overlapping cDNA clones (FIG. 4) wereidentified which show high homology to the human TIP-2 partial cDNA(GenBank accession number: AF028824) (Rousset, R., et al., Oncogene 16(1998) 643-654) and were designated as IIP-1a and IIP-1b. The IIP-1acDNA corresponds to nt 117 to 751 of TIP-2. The IIP-1b cDNA showsbesides TIP-2 sequences (nt 1 to 106) additional 5′ sequences which arehomologous to sequence Y2H35 of WO 97/27296 (nt 25 to 158).

IIP-1a and IIP-1b both share the sequence coding for the PDZ domain ofTIP-2 (nt 156 to 410) which is a known protein-protein interactiondomain (Ponting, C. P., et al., BioEssays 19 (1997) 469-479). Bydeletion analysis the PDZ domain was determined as the essential andsufficient IGF-1 receptor binding domain of IIP-1 (FIG. 4).

Further yeast two-hybrid analysis revealed that binding of the IIP-1protein to the IGF-1 receptor is specific for this receptor tyrosinekinase. No interaction was seen to the insulin receptor or Ros. Receptortyrosine kinases of other families did not interact with IIP-1 (e.g.Met, Ret, Kit, Fms, Neu, EGF receptor). Thus, IIP-1 most likely is thefirst interaction protein shown to be specific for the IGF-1 receptortyrosine kinase. IIP-1 also binds to the kinase inactive mutant of theIGF-1 receptor.

IIP-2

IIP-2 was identified as a new binder of the cytoplasmic part of theIGF-1 receptor which corresponds to human APS (EMBL accession number:HSAB520). APS has been previously isolated in a yeast two-hybrid screenusing the oncogenic c-kit kinase domain as bait (Yokouchi, M., et al.,Oncogene 15 (1998) 7-15). IIP-2 interacts with the IGF-1 receptor in akinase dependent manner. Binding of IIP-2 was also observed to othermembers of the insulin receptor family (insulin receptor, Ros), but notto an unrelated receptor tyrosine kinase (Met). The region of IIP-2which was found to interact with the IGF-1 receptor corresponds to humanAPS (nt 1126 to 1674, EMBL Acc No. AB000520) contains the SH2 domain ofAPS (nt 1249 to 1545).

IIP-3

IIP-3 was isolated as a new IGF-1 receptor interacting protein and isidentical to PSM (GenBank accession number: AF020526). PSM is known as aPH and SH2 domain containing signal transduction protein which binds tothe activated insulin receptor (Riedel, H., et al., J. Biochem. 122(1997) 1105-1113). A variant of PSM has also been described (Riedel, H.,et al., J. Biochem. 122 (1997) 1105-1113). Binding of IIP-3 to the IGF-1receptor is dependent on tyrosyl phosphorylation of the receptor.

A cDNA clone corresponding to nt 1862 to 2184 of the variant form of PSMwas identified. The isolated cDNA clone turned out to code for the IGF-1receptor binding region. The SH2 domain of PSM (nt 1864 to 2148, EMBLAcc No. AF020526) is encoded by the sequence of the IIP-3 partial cDNAclone isolated.

IIP-4

IIP-4 was isolated as a new interacting protein of the cytoplasmicdomain of the IGF-1 receptor. IIP-4 corresponds to p59fyn, a src-liketyrosine kinase (EMBL accession number: MMU70324 and human fynEM_HUM1:HS66H14) (Cooke, M. P., and Perlmutter, R. M., New Biol. 1(1989) 66-74). IIP-4 binds in a kinase dependent manner to the IGF-1receptor and to several other receptor tyrosine kinases as to theinsulin receptor and Met. The region of IIP-4 interacting with the IGF-1receptor (nt 665 to 1044) contains the SH2 domain of p59fyn (EMBL AccNo. U70324).

IIP-5

IIP-5 was isolated as a new IGF-1 receptor interacting protein. IIP-5shows a high homology to the zinc finger protein Zfp38 (EMBL accessionnumber: MMZFPTA) and is at least 80% homologous to the correspondinghuman gene. Zfp-38 is known as a transcription factor (Chowdhury, K., etal., Mech. Dev. 39 (1992) 129-142). IIP-5 interacts exclusively with theactivated and phosphorylated IGF-1 receptor, but not with a kinaseinactive mutant. In addition to binding of IIP-5 to the IGF-1 receptorinteraction of IIP-5 with receptor tyrosine kinases of the insulinreceptor family (insulin receptor, Ros) was observed. IIP-5 does notbind to the more distantly related receptor tyrosine kinase Met.

One cDNA clone binding to the IGF-1 receptor which codes for nt 756 to1194 of Zfp38 (EMBL Acc No. MMZFPTA) and contains the first zinc finger(nt 1075 to 1158) was isolated. This domain is sufficient for binding tothe activated IGF-1 receptor.

IIP-6

IIP-6 was identified as a new IGF-1 receptor interacting protein. IIP-6shows weak similarity to the zinc finger domain of Zfp29 (EMBL accessionnumber: MMZFP29). Zfp29 consists of a N-terminal transcriptionalactivation domain and 14 C-terminal Cys²His² zinc fingers (Denny, P.,and Ashworth, A., Gene 106 (1991) 221-227). Binding of IIP-6 to theIGF-1 receptor depends on phosphorylation of the IGF-1 receptor kinase.IIP-6 also binds to the insulin receptor, but does not interact withMet. The region of IIP-6 found to interact with the IGF-1 receptor (SEQID NO:3, SEQ ID NO:4) contains two zinc finger domains of the Cys²His²type.

IIP-7

IIP-7 was isolated as a new IGF-1 receptor interacting protein whichcorresponds to Pax-3 (EMBL accession number: MMPAX3R and human Pax3EM-HUM2:S69369). Pax-3 is known as a DNA-binding protein being expressedduring early embryogenesis (Goulding, M. D., et al., EMBO J. 10 (1991)1135-1147). IIP-7 binds in a phosphorylation dependent manner to theIGF-1 receptor. IIP-7 also interacts with the insulin receptor and Met.A partial IIP-7 cDNA clone turned out to code for the IGF-1 receptorbinding domain of Pax3 (nt 815 to 1199, EMBL Acc No. MMPAX3R). Thisregion contains the Pax-3 paired domain octapeptide (nt 853 to 876) andthe paired-type homeodomain (nt 952 to 1134).

IIP-8

IIP-8 codes for the full-length cDNA of Grb7 (EMBL accession number:MMGRB7P, human Grb7 EM_HUM1:AB008789). Grb7, a PH domain and a SH3domain containing signal transduction protein, was first published as anEGF receptor-binding protein (Margolis, B. L., et al., Proc. Natl. Acad.Sci. USA 89 (1992) 8894-8898). IIP-8 does not interact with the kinaseinactive mutant of the IGF-1 receptor. Binding of IIP-8 to several otherreceptor tyrosine kinases (e.g. insulin receptor, Ros and Met) was alsoobserved.

IIP-9

IIP-9 was identified as a new IGF-1 receptor interaction protein. IIP-9is identical to nck-beta (EMBL Acc No. AF043260). Nck is a cytoplasmicsignal transduction protein consisting of SH2 and SH3 domains (Lehmann,J. M., et al., Nucleic Acids Res. 18 (1990) 1048). IIP-9 interacts withthe IGF-1 receptor in a phosphorylation dependent manner. nck binds tothe juxtamembrane region of the IGF-1 receptor. Apart from binding ofIIP-9 to the IGF-1 receptor, interaction with the insulin receptor butnot with Ros or Met was seen.

A preferred object of the invention are polypeptides that arehomologous, and more preferably, polypeptides that are substantiallyidentical to the polypeptides of SEQ ID NO:6 (IIP-10). Homology can beexamined by using the FastA algorithm described by Pearson, W. R.,Methods in Enzymology 183 (1990) 63-68, Academic Press, San Diego, US.By “substantially identical” is meant an amino acid sequence whichdiffers only by conservative amino acid substitutions, for examplesubstitutions of one amino acid for another of the same class (e.g.valine for glycine, arginine for lysine, etc.) or by one or morenon-conservative amino acid substitution, deletions or insertionslocated at positions of the amino acid sequence which do not destroy thebiological function of the polypeptide. This includes substitution ofalternative covalent peptide bonds in the polypeptide. By “polypeptide”is meant any chain of amino acids regardless of length orposttranslational modification (e.g., glycosylation or phosphorylation)and can be used interchangeably with the term “protein”.

According to the invention by “biologically active fragment” is meant afragment which can exert a physiological effect of the full-lengthnaturally-occurring protein (e.g., binding to its biological substrate,causing an antigenic response, etc.).

The invention also features fragments of the polypeptide according tothe invention which are antigenic. The term “antigenic” as used hereinrefers to fragments of the protein which can induce a specificimmunogenic response, e.g. an immunogenic response which yieldsantibodies which specifically bind to the protein according to theinvention. The fragments are preferably at least 8 amino acids, andpreferably up to 25 amino acids, in length. In one preferred embodiment,the fragments include the domain which is responsible for the binding ofthe IIPs to the IGF-1 receptor (i.e., the PDZ domain of IIP-1. By“domain” is meant the region of amino acids in a protein directlyinvolved in the interaction with its binding partner. PDZ domains areapproximately 90-residue repeats found in a number of proteinsimplicated in ion-, channel and receptor clustering and the linking ofreceptors to effector enzymes. Such PDZ are described in general byCabral, J. H., et al., Nature 382 (1996) 649-652.

The invention further comprises a method for producing a proteinaccording to the invention whose expression or activation is correlatedwith tumor proliferation, by expressing an exogenous DNA in prokaryoticor eukaryotic host cells and isolation of the desired protein orexpressing said exogeneous DNA in vivo for pharmaceutical means, whereinthe protein is coded preferably by a DNA sequence coding for IIP-10,preferably the DNA sequence shown in SEQ ID NO:5.

The polypeptides according to the invention can also be produced byrecombinant means, or synthetically. Non-glycosylated IIP-10 polypeptideis obtained when it is produced recombinantly in prokaryotes. With theaid of the nucleic acid sequences provided by the invention it ispossible to search for the IIP-10 gene or its variants in genomes of anydesired cells (e.g. apart from human cells, also in cells of othermammals), to identify these and to isolate the desired gene coding forthe IIP-10 protein. Such processes and suitable hybridization conditions(see also above, “stringent conditions”) are known to a person skilledin the art and are described, for example, by Sambrook et al., MolecularCloning: A laboratory manual (1989) Cold Spring Harbor Laboratory Press,New York, USA, and Hames, B. D., Higgins, S. G., Nucleic acidhybridisation—a practical approach (1985) IRL Press, Oxford, England. Inthis case the standard protocols described in these publications areusually used for the experiments.

The use of recombinant DNA technology enables the production of numerousactive IIP-10 derivatives. Such derivatives can, for example, bemodified in individual or several amino acids by substitution, deletionor addition. The derivatization can, for example, be carried out bymeans of site directed mutagenesis. Such variations can be easilycarried out by a person skilled in the art (J. Sambrook, B. D. Hames,loc. cit.). It merely has to be ensured by means of the below-mentionedtumor cell growth inhibition assay that the characteristic properties ofIIP-10 are preserved.

With the aid of such nucleic acids coding for an IIP-10 protein, theprotein according to the invention can be obtained in a reproduciblemanner and in large amounts. For expression in prokaryotic or eukaryoticorganisms, such as prokaryotic host cells or eukaryotic host cells, thenucleic acid is integrated into suitable expression vectors, accordingto methods familiar to a person skilled in the art. Such an expressionvector preferably contains a regulatable/inducible promoter. Theserecombinant vectors are then introduced for the expression into suitablehost cells such as, e.g., E. coli as a prokaryotic host cell orSaccharomyces cerevisiae, teratocarcinoma cell line PA-1 sc 9117(Büttner et al., Mol. Cell. Biol. 11 (1991) 3573-3583), insect cells,CHO or COS cells as eukaryotic host cells and the transformed ortransduced host cells are cultured under conditions which allowexpression of the heterologous gene. The isolation of the protein can becarried out according to known methods from the host cell or from theculture supernatant of the host cell. Such methods are described forexample by Ausubel I., Frederick M., Current Protocols in Mol. Biol.(1992), John Wiley and Sons, New York. Also in vitro reactivation of theprotein may be necessary if it is not found in soluble form in the cellculture.

The invention therefore in addition concerns a IIP polypeptide which isa product of prokaryotic or eukaryotic expression of an exogenous DNA.

The protein can be isolated from the cells or the culture supernatantand purified by chromatographic means, preferably by ion exchangechromatography, affinity chromatography and/or reverse phase HPLC.

IIP-10 can be purified after recombinant production by affinitychromatography using known protein purification techniques, includingimmunoprecipitation, gel filtration, ion exchange chromatography,chromatofocusing, isoelectric focussing, selective precipitation,electrophoresis, or the like.

Diagnostic Methods:

The invention further comprises a method for detecting a nucleic acidmolecule encoding an IIP-gene, comprising incubating a sample (e.g.,body fluids such as blood, cell lysates) with a nucleic acid moleculeaccording to the invention and determining hybridization under stringentconditions of said nucleic acid molecule to a target nucleic acidmolecule for determination of presence of a nucleic acid molecule whichis said IIP gene and therefore a method for the identification of IGF-1Ractivation or inhibition in mammalian cells or body fluids.

Therefore the invention also includes a method for the detection of theproliferation potential of a tumor cell comprising

a) incubating a sample of body fluid of a patient suffering from cancer,a sample of cancer cells, or a sample of a cell extract or a cellculture supernatant of said cancer cells, whereby said sample containsnucleic acids with a nucleic acid probe which is selected from the groupconsisting of

-   -   (i) the nucleic acids shown in SEQ ID NOS:1, 3 or 5 or a nucleic        acid which is complementary thereto and    -   (ii) nucleic acids which hybridize under stringent conditions        with one of the nucleic acids from (i) and

b) detecting hybridization by means of a further binding partner of thenucleic acid of the sample and/or the nucleic acid probe or by X-rayradiography.

Hybridization between the probe and nucleic acids from the sampleindicates the presence of the RNA of such proteins. Such methods areknown to a person skilled in the art and are described, for example, inWO 89/06698, EP-A 0 200 362, U.S. Pat. No. 2,915,082, EP-A 0 063 879,EP-A 0 173 251, EP-A 0 128 018.

In a preferred embodiment of the invention the coding nucleic acid ofthe sample is amplified before the test, for example by means of theknown PCR technique. Usually a derivatized (labeled) nucleic acid probeis used within the framework of nucleic acid diagnostics. This probe iscontacted with a denatured DNA or RNA from the sample which is bound toa carrier and in this process the temperature, ionic strength, pH andother buffer conditions are selected—depending on the length andcomposition of the nucleic acid probe and the resulting meltingtemperature of the expected hybrid—such that the labeled DNA or RNA canbind to homologous DNA or RNA (hybridization see also Wahl, G. M., etal., Proc. Natl. Acad. Sci. USA 76 (1979) 3683-3687). Suitable carriersare membranes or carrier materials based on nitrocellulose (e.g.,Schleicher and Schüll, BA 85, Amersham Hybond, C.), strengthened orbound nitrocellulose in powder form or nylon membranes derivatized withvarious functional groups (e.g., nitro groups) (e.g., Schleicher andSchüll, Nytran; NEN, Gene Screen; Amersham Hybond M.; Pall Biodyne).

Hybridizing DNA or RNA is then detected by incubating the carrier withan antibody or antibody fragment after thorough washing and saturationto prevent unspecific binding. The antibody or the antibody fragment isdirected towards the substance incorporated during derivatization intothe nucleic acid probe. The antibody is in turn labeled. However, it isalso possible to use a directly labeled DNA. After incubation with theantibodies it is washed again in order to only detect specifically boundantibody conjugates. The determination is then carried out according toknown methods by means of the label on the antibody or the antibodyfragment.

The detection of the expression can be carried out for example as:

in situ hybridization with fixed whole cells, with fixed tissue smearsand isolated metaphase chromosomes,

colony hybridization (cells) and plaque hybridization (phages andviruses),

Southern hybridization (DNA detection),

Northern hybridization (RNA detection),

serum analysis (e.g., cell type analysis of cells in the serum byslot-blot analysis),

after amplification (e.g., PCR technique).

Preferably the nucleic acid probe is incubated with the nucleic acid ofthe sample and the hybridization is detected optionally by means of afurther binding partner for the nucleic acid of the sample and/or thenucleic acid probe.

The nucleic acids according to the invention are hence valuableprognostic markers in the diagnosis of the metastatic and progressionpotential of tumor cells of a patient.

Screening for Antagonists and Agonists of IIPs or Inhibitors

According to the invention antagonists of IIP-10 or inhibitors for theexpression of IIP (e.g., antisense nucleic acids) can be used to inhibittumor progression and cause massive apoptosis of tumor cells in vivo,preferably by somatic gene therapy.

Therefore, the present invention also relates to methods of screeningfor potential therapeutics for cancer, diabetes, neurodegenerativedisorders, bone diseases, to methods of treatment for disease and tocell lines and animal models useful in screening for and evaluatingpotentially useful therapies for such disease. Therefore another objectof the invention are methods for identifying compounds which haveutility in the treatment of the afore-mentioned and related disorders.These methods include methods for modulating the expression of thepolypeptides according to the invention, methods for identifyingcompounds which can selectively bind to the proteins according to theinvention and methods of identifying compounds which can modulate theactivity of said polypeptides. These methods may be conducted in vitroand in vivo and may employ the transformed cell lines and transgenicanimal models of the invention.

An antagonist of IIPs or an inhibitor of IIP is defined as a substanceor compound which inhibits the interaction between IGF-1R and IIP,preferably IIP-10. Therefore the biological activity of IGF-1R decreasesin the presence of such a compound. In general, screening procedures forIIP antagonists involve contacting candidate substances with IIP-bearinghost cells under conditions favorable for binding and measuring theextent of decreasing receptor mediated signaling (in the case of anantagonist). Such an antagonist is useful as a pharmaceutical agent foruse in tumor therapy. For the treatment of diabetes, neural diseases, orbone diseases, stimulation of the signaling pathway is required, i.e.,screening for agonists is useful.

IIP activation may be measured in several ways. Typically, theactivation is apparent by a change in cell physiology such as anincrease or decrease in growth rate or by a change in thedifferentiation state or by a change in cell metabolism which can bedetected in standard cell assays, for example MTT or XTT assays (RocheDiagnostics GmbH, DE).

The nucleic acids and proteins according to the invention couldtherefore also be used to identify and design drugs which interfere withthe interaction of IGF-1R and IIPs. For instance, a drug that interactswith one of the proteins could preferentially bind it instead ofallowing binding its natural counterpart. Any drug which could bind tothe IGF-1 receptor and, thereby, prevent binding of an IIP or, viceversa, bind to an IIP and, thereby, prevent binding of the IGF-1receptor. In both cases, signal transduction of the IGF-1 receptorsystem would be modulated (preferably inhibited). Screening drugs forthis facility occurs by establishing a competitive assay (assay standardin the art) between the test compound and interaction of IIP and theIGF-1 receptor and using purified protein or fragments with the sameproperties as the binding partners.

The protein according to the invention is suitable for use in an assayprocedure for the identification of compounds which modulate theactivity of the proteins according to the invention. Modulating theactivity as described herein includes the inhibition or activation ofthe protein and includes directly or indirectly affecting the normalregulation of said protein activity. Compounds which modulate theprotein activity include agonists, antagonists and compounds whichdirectly or indirectly affect the regulation of the activity of theprotein according to the invention. The protein according to theinvention may be obtained from both native and recombinant sources foruse in an assay procedure to identify modulators. In general, an assayprocedure to identify modulators will contain the IGF receptor, aprotein of the present invention, and a test compound or sample whichcontains a putative modulator of said protein activity. The testcompounds or samples may be tested directly on, for example, purifiedprotein of the invention, whether native or recombinant, subcellularfractions of cells producing said protein, whether native orrecombinant, and/or whole cells expressing said protein, whether nativeor recombinant. The test compound or sample may be added to the proteinaccording to the invention in the presence or absence of knownmodulators of said protein. The modulating activity of the test compoundor sample may be determined by, for example, analyzing the ability ofthe test compound or sample to bind to said protein, activate saidprotein, inhibit its activity, inhibit or enhance the binding of othercompounds to said protein, modifying receptor regulation or modifyingintracellular activity.

The identification of modulators of the protein activity are useful intreating disease states involving the protein activity. Other compoundsmay be useful for stimulating or inhibiting the activity of the proteinaccording to the invention. Such compounds could be of use in thetreatment of diseases in which activation or inactivation of the proteinaccording to the invention results in either cellular proliferation,cell death, non-proliferation, induction of cellular neoplastictransformations, or metastatic tumor growth and hence could be used inthe prevention and/or treatment of cancers such as, for example,prostate and breast cancer. The isolation and purification of a DNAmolecule encoding the protein according to the invention would be usefulfor establishing the tissue distribution of said protein as well asestablishing a process for identifying compounds which modulate theactivity of said protein and/or its expression.

Therefore a further embodiment of the invention is a method forscreening a compound that inhibits the interaction between IGF-1R andIIP-1, IIP-2, IIP3, IIP4, IIP5, IIP6, IIP7, IIP8, IIP9 or IIP-10,comprising

a) combining IGF-1R and the IIP polypeptide with a solution containing acandidate compound such that the IGF-1R and the IIP polypeptide arecapable of forming a complex andb) determining the amount of complex relative to the predetermined levelof binding in the absence of said candidate compound and therefromevaluating the ability of said candidate compound to inhibit binding ofIGF-1R to the IIP polypeptide.

Such a screening assay is preferably performed as an ELISA assay wherebyIGF-1R or the IIP-polypeptide preferably IIP-1 or IIP-10, is bound on asolid phase. A further embodiment of the invention is a method for theproduction of a therapeutic agent for the treatment of carcinomas in apatient comprising combining a therapeutically effective amount of acompound which inhibits the interaction between IGF-1R and IIP inbiochemical and/or cellular assays to an extent of at least 50%.Biochemical assays are preferably ELISA-based assays or homogeneousassays. In the case of the ELISA system antibodies specific for the twobinding partners are used for detection of the complexes. In the case ofthe homogenous assay at least one binding partner is labeled withfluorophores which allows analysis of the complexes. Cellular assays arepreferably assays whereby tumor cells or cells transfected withexpression constructs of the IGF-1R and the respective binding proteinsare treated with or without drugs and complex formation between the twocomponents is then analyzed using standard cell assays.

A preferred embodiment of the invention is a method for the productionof a therapeutic agent for the treatment of carcinomas in a patientcomprising combining a pharmaceutically acceptable carrier with atherapeutically effective amount of a compound which inhibits theinteraction between IGF-1R and an IIP-polypeptide, preferably IIP-1 orIIP-10, in a cellular assay, whereby in said cellular assay tumor cellsor cells transfected with expression constructs of IGF-1R and of therespective IIP are treated with said compound, and complex formationbetween IGF-1R and said respective IIP is analyzed, and the extent ofsaid complex formation in the case of inhibition does not exceed 50%referred to 100% for complex formation without said compound in saidsame cellular assay.

A further embodiment of the invention is a method of treating a patientsuffering from a carcinoma with a therapeutically effective amount of acompound which inhibits the interaction between IGF-1R and theIIP-polypeptide, preferably IIP-1 or IIP-10, in a cellular assay,whereby in said cellular assay tumor cells or cells transfected withexpression constructs of IGF-1R and of the respective IIP are treatedwith said compound, and complex formation between IGF-1R and saidrespective IIP is analyzed, and the extent of said complex formation inthe case of inhibition does not exceed 50% referred to 100% for complexformation without said compound in said same cellular assay.

A further embodiment of the invention is an antibody against IIP-1 orIIP-10 according to the invention.

Antibodies were generated from the human, mouse, or rat polypeptides.Antibodies specifically recognizing IIP-1 or IIP-10 are encompassed bythe invention. Such antibodies are raised using standard immunologicaltechniques. Antibodies may be polyclonal or monoclonal or may beproduced recombinantly such as for a humanized antibody. An antibodyfragment which retains the ability to interact with IIP-1 or IIP-10 isalso provided. Such a fragment can be produced by proteolytic cleavageof a full-length antibody or produced by recombinant DNA procedures.Antibodies of the invention are useful in diagnostic and therapeuticapplications. They are used to detect and quantitate IIP-1 or IIP-10 inbiological samples, particularly tissue samples and body fluids. Theyare also used to modulate the activity of IIP-1 or IIP-10 by acting asan agonist or an antagonist.

The following examples, references, sequence listing and drawing areprovided to aid the understanding of the present invention, the truescope of which is set forth in the appended claims. It is understoodthat modifications can be made in the procedures set forth withoutdeparting from the spirit of the invention.

EXAMPLES Example 1 Isolation and Characterization of IGF-1R BindingProteins

The yeast two-hybrid system (Fields, S., and Song, O., Nature 340 (1989)245-246) was used to isolate unknown cytosolic IGF-1 receptor bindingproteins. For screening a modified version of the yeast two-hybridsystem was used which allows interchain tyrosylphosphorylation of thereceptors in yeast.

The yeast two-hybrid bait plasmid (BTM116-cpIGF-1 receptor) wasconstructed by fusing the cytoplasmic domain of the β-subunit of theIGF-1 receptor (nt 2923 to 4154) (Ullrich, A., et al., EMBO J. 5 (1986)2503-2512) to the LexA DNA-binding domain which forms dimers and mimicsthe situation of the activated wildtype receptor (cf. Weidner, M., etal., Nature 384 (1996) 173-176). By introducing a proline-glycine spacerbetween the LexA DNA-binding domain and the receptor domain the abilityof the bait to bind known substrates of the IGF-1 receptor wasremarkably increased in comparison to other spacer amino acids (FIG. 1).

Alternatively a bait was constructed containing only the juxtamembraneor C-terminal region of the IGF-1 receptor (nt 2923 to 3051 or nt 3823to 4146) (Ullrich, A., et al., EMBO J. 5 (1986) 2503-2512) fused to thekinase domain of an unrelated, very potential receptor tyrosine kinase.Here the kinase domain of tpr met (nt 3456 to 4229) (GenBank accessionnumber: HSU19348) (FIG. 2) was used. In this way it is possible todelineate the region of the IGF-1 receptor which mediates binding todownstream effectors.

The IGF-1 receptor bait plasmid was used to screen activation domaincDNA libraries (e.g. VP16- or Gal4 based activation domain) (cf.Weidner, M., et al., Nature 384 (1996) 173-176). The bait and preyplasmids were co-transfected into Saccharomyces cerevisiae strain L40containing a HIS3 and lacZ reporter gene. Library plasmids were isolatedfrom yeast colonies growing on histidine deficient medium, weresequenced and reintroduced into yeast strain L40. By co-transfectingexperiments with different test baits, i.e. BTM116 plasmids coding for akinase inactive mutant of the IGF-1 receptor (L1033A) or the cytoplasmicdomain of receptor tyrosine kinases of the insulin receptor family(insulin receptor, Ros) and of unrelated receptor tyrosine kinasefamilies (Met, EGF receptor, Kit, Fms, Neu) the specificity of theputative bait-prey interactions was evaluated. Several cDNAs wereidentified which code for previously unknown IGF-1 receptor interactingproteins (IIPs). In addition binding domains of known substrates of theIGF-1 receptor such as the C-terminal SH2 domain of p85PI3K and the SH2domain of Grb 10 were found. The results are shown in Table 1.

TABLE 1 IIP wt IGF-1R mu IGF-1R IR Ros Met IIP-1 + + − − − IIP-2 + − + +− IIP-3 + − + + + IIP-4 + − + nd + IIP-5 + − + + − IIP-6 + − + nd −IIP-7 + − + nd + IIP-8 + − + + + IIP-9 + − + − − IIP-10 + − − nd Nd

Delineation of the binding specificity of the IIPs with respect todifferent receptor tyrosine kinases tested in the yeast two-hybridsystem. Yeast cells were cotransfected with a LexA fusion constructcoding for the different receptor tyrosine kinases and an activationplasmid coding for the different IIPs fused to the VP16 activationdomain. Interaction between the IIPs and the different receptor tyrosinekinases was analyzed by monitoring growth of yeast transfectants platedout on histidine deficient medium and incubated for 3d at 30° C. (wtIGF-1R, kinase active IGF-1 receptor; mu IGF-1 R, kinase inactive mutantIGF-1 receptor; IR, insulin receptor; Ros, Ros receptor tyrosine kinase;Met, Met receptor tyrosine kinase; +, growth of yeast transfectantswithin 3 days larger than 1 mm in diameter; −, no detected growth; nd,not determined).

Example 2 Assay Systems A) In-Vitro/Biochemical Assays:

ELISA-Based Assay/Homogenous Assay

IGF-1R and the binding proteins (IIPs) are expressed with or withoutTag-epitopes in E. coli or eucaryotic cells and purified to homogeneity.Interaction of IGF-1R and the respective binding proteins are analyzedin the presence or absence of drugs. Compounds which either inhibit orpromote binding of IGF-1R and the respective binding proteins areselected. In the case of the ELISA system antibodies specific for thetwo binding partners are used for detection of the complexes. In thecase of the homogenous assay at least one binding partner is labeledwith fluorophores which allows analysis of the complexes. Alternatively,anti-Tag-antibodies are used to monitor interaction.

B) Cellular Assays:

Tumor cells or cells transfected with expression constructs of theIGF-1R and the respective binding proteins are treated with or withoutdrugs and complex formation between the two components is then analyzedusing standard assays.

Example 3 cDNA Cloning of IIP-1 and IIP-10 And RT-PCR-Assay

The nucleotide sequence of full length IIP-1 was determined bysequencing of the partial cDNA clones of IIP-1 and (IIP-1a, IIP-1b) andby using database information (ESTs). cDNA cloning of full length IIP-1was performed by RT PCR on total RNA isolated from a MCF7_(ADR)breastcell line. PT PCR with two oligonucleotide primers: TIP2c-s (SEQ IDNO:7) and TIP2b-r (SEQ ID NO:8) resulted in amplification of two DNAfragments of 1.0 kb (IIP-1) and 0.7 kb (IIP-1(p26)).

The nucleotide sequence of full length IIP-10 was determined bysequencing of the partial cDNA clone of IIP-10 and by using databaseinformation (ESTs). cDNA cloning of IIP-10 was performed on total RNAisolated from the colon cancer cell line SW480. RT PCR with twooligonucleotide primers: Hethyl-s (SEQ ID NO:9) and Hethyl-r (SEQ IDNO:10) resulted in amplification of a cDNA fragment of 676bp (IIP-10).

DNA sequencing was performed using the dideoxynucleotide chaintermination method on an ABI 373A sequencer using the Ampli Taq® FSDideoxyterminator kit (Perkin Elmer, Foster City, Calif.). Comparison ofthe cDNA and deduced protein sequences was performed using AdvancedBlast Search (Altschul, S. F., et al., J. Mol. Biol. 215 (1990) 403-410;Altschul, S. F., et al., Nucleic Acids Res. 25 (1997) 3389-3402).

Example 4 Western Blot Analysis of IIP-1 and IIP-10

Total cell lysates were prepared in a buffer containing 50 mM Tris pH8.0, 150 mM NaCl, 1% NP40, 0.5% deoxycholic acid, 0.1% SDS, and 1 mMEDTA and cleared by centrifugation for 15 min at 4° C. The proteinconcentration of the supernatants was measured using the Micro BCAProtein Assay kit (Pierce Chemical Co., Rockford, Ill.) according to themanufacturer's manual. IGF-1 receptors were immunoprecipitated usinganti-IGF-1 receptor antibodies (Santa Cruz). Proteins were fractionatedby SDS-PAGE and electrophoretically transferred to nitrocellulosefilters. Nitrocellulose filters were preincubated with 10% (w/v)fat-free milk powder in 20 mM Tris pH 7.5, 150 mM NaCl, 0.2% Tween-20.Binding of a mouse monoclonal antibody directed against the flag epitopewas detected by horseradish peroxidase-labeled goat-anti-mouse IgGantiserum (Biorad, Munich, DE) and visualized using an enhancedchemoluminescence detection system, ECL™ (Amersham, Braunschweig, DE).

Example 5 Overexpression of IIP-1 to IIP-10 in Mammalian Cells byLiposome-Mediated Transfection

The cDNAs for IIP-1 to -10 were cloned into the NotI site of pBATflag orpcDNA3flag (Weidner, K. M., et al., Nature 384 (1996) 173-176); Behrens,J., et al., Nature 382 (1996) 638-642; Behrens, J., et al., Science 280(1998) 596-599). NIH3T3 cells or other recipient cells were transfectedwith pcDNAflagIIP-1 to -10 or alternatively with pBATflag IIP-1 to -10using FuGENE6 (Roche Biochemicals) as transfection agent. Cells wereselected in 0.4 mg/ml G418. Single clones were picked and analyzed forexpression of IIP-1 to -10 and functionally characterized with respectto proliferation.

Northern Blot Analysis

Human and murine mRNA multiple tissue Northern blots were purchased fromClontech (Palo Alto, Calif., US). A cDNA probe spanning IIP-10nt343-nt676 of the coding region was labeled with DIG-dUTP using the PCRDIG Labeling Mix (Roche Diagnostics GmbH, DE). A digoxygenin labeledactin RNA probe was purchased from Roche Diagnostics GmbH, DE.Hybridization was performed using the DIG EasyHyb hybridization solution(Roche Diagnostics GmbH, DE). IIP-10 mRNA was detected with DIG-specificantibodies conjugated to alkaline phosphatase and the CSPD substrate(Roche Diagnostics GmbH, DE).

Example 6 Detection of mRNA in Cancer Cells

In order to detect whether proteins are expressed in cancer cells whichare coded by nucleic acids which hybridize with SEQ ID NO:1 or SEQ IDNO:5 or the complementary sequence and consequently whether mRNA ispresent, it is possible on the one hand to carry out the establishedmethods of nucleic acid hybridization such as Northern hybridization,in-situ hybridization, dot or slot hybridization and diagnostictechniques derived therefrom (Sambrook et al., Molecular Cloning: Alaboratory manual (1989) Cold Spring Harbor Laboratory Press, New York,USA; Hames, B. D., Higgins, S. G., Nucleic acid hybridisation—apractical approach (1985) IRL Press, Oxford, England; WO 89/06698; EP-A0 200 362; EP-A 0 063 879; EP-A 0 173 251; EP-A 0 128 018). On the otherhand it is possible to use methods from the diverse repertoire ofamplification techniques using specific primers (PCR Protocols—A Guideto Methods and Applications (1990), publ. M. A. Innis, D. H. Gelfand, J.J. Sninsky, T. J. White, Academic Press Inc.; PCR—A Practical Approach(1991), publ. M. J. McPherson, P. Quirke, G. R. Taylor, IRL Press).

The RNA for this is isolated from the cancer tissue by the method ofChomcszynski and Sacchi, Anal Biochem. 162 (1987) 156-159. 20 μg totalRNA was separated on a 1.2% agarose formaldehyde gel and transferredonto nylon membranes (Amersham, Braunschweig, DE) by standard methods(Sambrook et al., Molecular Cloning: A laboratory manual (1989) ColdSpring Harbor Laboratory Press, New York, USA. The DNA sequence SEQ IDNO:1 or SEQ ID NO:5 was radioactively labeled as probes (Feinberg, A.P., and Vogelstein, B., Anal Biochem. 137 (1984) 266-267). Thehybridization was carried out at 68° C. in 5×SSC, 5×Denhardt, 7% SDS/0.5M phosphate buffer pH 7.0, 10% dextran sulfate and 100 μg/ml salmonsperm DNA. Subsequently the membranes were washed twice for one houreach time in 1×SSC at 68° C. and then exposed to X-ray film.

Example 7 Procedure for Identification of Modulators of the Activity ofthe Protein According to the Invention

The expression vector of Example 5 (either for IIP-1 or IIP-10 10 μg/10⁶cells) is transferred into NIH 3T3 cells by standard methods known inthe art (Sambrook et al.). Cells which have taken up the vector areidentified by their ability to grow in the presence of the selection orunder selective conditions (0.4 mg/ml G418). Cells which express DNAencoding IIP produce RNA which is detected by Northern blot analysis asdescribed in Example 5. Alternatively, cells expressing the protein areidentified by identification of the protein by Western blot analysisusing the antibodies described in Example 4. Cells which express theprotein from the expression vector will display an altered morphologyand/or enhanced growth properties.

Cells which express the protein and display one or more of the alteredproperties described above are cultured with and without a putativemodulator compound. By screening of chemical and natural libraries, suchcompounds can be identified using high throughput cellular assaysmonitoring cell growth (cell proliferation assays using as chromogenicsubstrates the tetrazolium salts WST-1, MTT, or XTT, or a cell deathdetection ELISA using bromodesoxyuridine (BrdU); cf. Boehringer MannheimGmbH, Apoptosis and Cell Proliferation, 2^(nd) edition, 1998, pp.70-84).

The modulator compound will cause an increase or a decrease in thecellular response to the IIP protein activity and will be either anactivator or an inhibitor of IGF-receptor function, respectively.

Alternatively, putative modulators are added to cultures of tumor cells,and the cells display an altered morphology and/or display reduced orenhanced growth properties. A putative modulator compound is added tothe cells with and without IIP protein and a cellular response ismeasured by direct observation of morphological characteristics of thecells and/or the cells are monitored for their growth properties. Themodulator compound will cause an increase or a decrease in the cellularresponse to IIP protein and will be either an activator or an inhibitorof IGF-1 receptor activity, respectively.

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1. An isolated recombinant polypeptide, wherein the isolated recombinantpolypeptide (a) comprises an amino acid sequence that is a variant ofSEQ ID NO:6 that is at least 90% identical to SEQ ID NO:6 and (b) bindsto a human IGF-1 receptor.